Semaphorin pathways have been well characterized for their roles in axon guidance. Moreover, recent findings highlight significant roles of this pathway in other critical areas such as vasculogenesis, angiogenesis, cell migration/differentiation, immune regulation, and cancer pathology. The Pi's laboratory has recently identified zebra fish olfactomedin 2 (OM2), a secreted glycoprotein with a highly conserved olfactomedin domain. Initial expression and functional characterization studies revealed OM2's roles in three important developmental processes: axon guidance, neural crest migration/differentiation, and angiogenesis. Analyses on phenotypes of OM2 morphants in comparison with published phenotypes from disrupted semaphorin pathways, consistently led the PI to formulate the following overall hypothesis: OM2 regulates axon guidance, neural crest cell migration/differentiation, and angiogenesis via its interaction with the semaphorin pathway. In order to test the OM2-semaphorin pathway link in these processes, three Specific Aims are proposed. Specific Aim 1: To test the hypothesis that highly specific cranial axon guidance defects in OM2 morphants are due to the perturbation of direct interaction between OM2 and semaphorin receptor complexes. Specific Aim 2: To test the hypothesis that the absence of pharyngeal cartilages in OM2 morphants is due to the perturbation of cranial neural crest cell (cNCC) migration and/or differentiation, which is critically dependent upon semaphorin pathways. Specific Aim 3: To test the hypothesis that highly specific defects found only in late-onset cranial vasculature in OM2 morphants are due to perturbation in semaphorin-neuropilin and/or VEGF-neuropilin pathways. These hypotheses will be tested by (1) high resolution expression analysis of OM2 and semaphorin signaling molecules, deficiency of which results in highly similar phenotypes as OM2-deficiency phenotypes;(2) concomitant inhibition of semaphorin components and OM2 to verify functional convergence of OM2 in the semaphorin signaling pathway;(3) testing direct molecular interactions between OM2 and components of the semaphorin receptor complex. Given that the semaphorin pathway is known to be critically involved in adult nerve regeneration, craniofacial malformation (most frequent human birth defects), and tumor angiogenesis, novel insights into the regulation of this signaling pathway will be of paramount health benefit.